PROJECT SUMMARY Epigenetic plasticity within oral squamous cell carcinomas (OSCCs) limits the efficacy of targeting PI3K/Akt, the most mutated oncogenic pathway in this disease. We have shown that maximal Akt activation occurs in only a small subset of OSCC cells and depends on the H3K4 demethylase JARID1B. By contrast, PI3K signaling is suppressed in a quiescent cell fraction that is enriched by anti-PI3K drugs. These non-dividing G0- like cells are primed to upregulate JARID1B, which reprograms them into an Akt-hyperactive state that divides slowly and is sensitive to PI3K inhibition. The stem cell-like molecular profile and high tumorigenicity of these JARID1Bhigh cells implicate them as drivers of OSCC progression and intermediates for G0-like cells returning to rapid division. When JARID1Bhigh cells are depleted by PI3K inhibitors, the G0-like fraction expands and may sustain tumor growth by an alternate, JARID1B-independent pathway allowing their exit from quiescence. Our working hypothesis is that G0-like cells are driven by JARID1B into a PI3K-dependent, stem cell-like phenotype but also play a central role in sustaining OSCC growth under PI3K inhibition by bypassing this state. This proposal determines JARID1B's contribution to G0-like cells exiting quiescence by testing the hypothesis that JARID1B-mediated silencing of p27kip1 and PTEN allows PI3K hyper-activation to drive G0-like cells from quiescence to an aggressive phenotype (aim 1). It will also delineate the JARID1Bhigh state's mechanistic basis and direct role in OSCC progression. In this setting, JARID1B is hypothesized to enforce a slow cycling state with high invasive and metastatic potential by prolonging S/G2 and suppressing the miR-200 family (aim 2). The roles of G0-like and JARID1Bhigh cells will also be defined under PI3K inhibition, where JARID1Bhigh cells hypothetically return to a G0-like state that then sustains cancer progression via MAPK pathway compensation in a JARID1B-independent manner (aim3). Defining JARID1B's regulation of the tumor cell heterogeneity underlying PI3K/Akt activation offers a novel basis to treat PI3K-driven malignancy. Specifically, targeting JARID1B holds promise to both deplete aggressive, Akt-hyperactive states and allow combined inhibition of MAPK signals or other targets with low toxicity. This proposal thus helps realize the potential of therapies integrating current drugs with emerging tools to target epigenetic regulators.